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I'm working with a calcium assay to study the effects of different virulence factors. The assay works, but from day to day the signals of cell lysis go down. Unfortunately, I haven't found an explanation for why this is happening.

The particular assay I use measures cell lysis. The virulence factors I use cause pore formation and lead to a calcium influx (from the buffer). Esterase proteins activate the fluorophore and need calcium. Increase in cell lysis leads to an increase in signal.

As I already mentioned my signals are dropping (from day to day). But I don't know why.

Have you got an idea of what could lead to such effects?

Here are some factors that might be involved.

  • The virulence factors lose their activity? (I don't feel like that this causes the problem because new stocks also show a decrease in activity, and also the signal from the positive control decreases. I would say it decreases in a similar ratio. The positive control is triton.
  • The dye loses its activity? The bottle is normally stored in a freezer. I just take it out for the assay. Before I use it, I place the bottle on a shaker for about 30 minutes. I just open the bottle for a few seconds and then put it then back in the freezer.
  • The plate reader is broken or the reading protocol has changed? I already checked the protocol and every thing is the same as in the beginning. Coworkers get the same results with the same plate reader as before. So I guess, this should not be a problem.
  • The buffer gets destroyed from the procedure? I store it in the freezer and take it out and put in a waterbath. After I used it I place it back in the freezer. I carry out this procedure every time, can this lead to negative effects? The medium contains calcium, can something happen with it (Calcium plays an important role in this assay)? A colleague already told me that he stores his buffer in the fridge not in the freezer, and he didn't got such effects. But perhaps it's not the only thing he does different..
  • The cell number changes? The negative control does not change over the experiments, and the signal from it is still higher than the values from the blank. But i will check that.
  • Could my cells cause problems? They look still good under the microscope.
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  • $\begingroup$ Welcome to Biology.SE! Thank you for taking the tour, but please also go through the help center and Biology Meta pages starting with How to Ask questions effectively on this site and edit your question accordingly. In particular, please make sure your question is clear and as concise as possible (this includes spell and grammar checking). In addition, please don't include signatures etc. — your post has your ID attached and by posting you've already asked for help! Thanks! 😊 $\endgroup$ – tyersome Dec 16 '20 at 23:18
  • $\begingroup$ I've edited your post to correct language problems, but I suggest you add much more detail about exactly what fluorophore you are using (and in what solvent) and the constituents of the various other buffers you are using. Please check to make sure I've not changed your meaning. ——— I'll post a preliminary possible answer as well, but you probably need to get someone in your lab to teach you how to trouble shoot. IMO learning how to trouble shoot experiments is the most important lab skill! $\endgroup$ – tyersome Dec 16 '20 at 23:35
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Freeze-thaw cycles are often suspected of causing degradation in organic molecules 1,2. My first guess would be that your fluorophore is breaking down due to those repeated cycles. Alternatively, you might be getting precipitation of a calcium compound from the buffer you are freezing that your colleague is not.

Standard laboratory practice is to make aliquots of frozen solutions. Before thawing the solution for the first time, set up pre-labeled smaller containers (e.g. Eppendorf tubes) and then aliquot the amount you expect to use for each experiment. That way potentially sensitive compounds are only thawed twice.

References:

1: Kozikowski, B. A., Burt, T. M., Tirey, D. A., Williams, L. E., Kuzmak, B. R., Stanton, D. T., ... & Nelson, S. L. (2003). The effect of freeze/thaw cycles on the stability of compounds in DMSO. Journal of biomolecular screening, 8(2), 210-215.

2: US Food and Drug Administration. (2008). Guidance for Industry. Drug Stability Guidelines.

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  • $\begingroup$ Hey MattDMo, thanks for your answer! Today I made a special test, to eleminate some of the possible factors which could lead to such mysterious effects. I seeded out some cells and instead of treating them with my virulance factors i treated them with triton X. By the way I also seeded out higher and lower cell numbers than normaly used in the assay. More Cells led to more fluorescence, but the effect was small. I found something! When I observed the cells afterwards with the microscope, I saw that they haven't been lysed. How can that happen? They incubated over an hour in triton X? $\endgroup$ – Mourinho_1 Dec 17 '20 at 19:27
  • $\begingroup$ I guess that the triton was fine because also the virulance factors showed a signal loss. At the moment I feel like that the cells have changed, dispite the fact that they look like they should. Perhaps I should mention here, that this culture was startet about 3 weeks ago, the Inocolum was stored in liquid nitrogen. No abnormalities. $\endgroup$ – Mourinho_1 Dec 17 '20 at 19:53
  • $\begingroup$ *The cellline was startet 3 weeks ago. I guess the cells have been splitted now for about the sixth time. $\endgroup$ – Mourinho_1 Dec 17 '20 at 20:04

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